The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 38  |  Issue : 1  |  Page : 17--22

Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100)


Deena M.M. Habashy1, Mohamed R. Mohamed2,  
1 Department of Clinical Pathology, Hematology Unit, Ain Shams University, Cairo, Egypt
2 Department of Opthalmology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence Address:
Deena M.M. Habashy
Department of Clinical Pathology, Hematology Unit, Faculty of Medicine, Ain Shams University, 11566 Cairo
Egypt

Abstract

Background

Platelet hyperactivity has been reported in diabetic patients. Some evidence suggests that platelet hyperaggregation may participate in the pathogenesis of diabetic complications such as retinopathy.

Objectives

We aimed to compare platelet aggregation (PA) in patients with type II diabetes with no apparent retinopathy (NAR), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR) patients, and healthy individuals to evaluate the possible role of PA in the pathogenesis and staging of DR.

Participants and methods

Blood samples from 30 patients (10 diabetics with NAR, 10 with NPDR, and 10 with PDR) and 10 healthy individuals were assayed for PA using a platelet function analyzer-100.

Results

On comparing all the studied groups in terms of the demographic, clinical, and laboratory parameters, the mean age and the duration of diabetes were significantly higher in the PDR group (P<0.001). The PDR group also showed higher levels of platelet count, glycated hemoglobin, and shorter closure time (CT) (P<0.001). Both NPDR and PDR showed higher levels of fibrinogen (P<0.001). CT was correlated inversely with the fibrinogen level in the NAR group (P=0.000) and with the duration of diabetes in the NPDR group (P=0.04). No correlation was found between CT and any of the parameters studied in the PDR group (P>0.05).

Conclusion

PA is increased in the PDR stage in comparison with NAR, NPDR stages, and healthy individuals. This may provide a clue of its role in the pathogenesis and staging of DR. Also, this study highlights the crucial contribution of glycemic control and duration of diabetes in the progression of DR.




How to cite this article:
Habashy DM, Mohamed MR. Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100).Egypt J Haematol 2013;38:17-22


How to cite this URL:
Habashy DM, Mohamed MR. Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100). Egypt J Haematol [serial online] 2013 [cited 2020 Jan 26 ];38:17-22
Available from: http://www.ehj.eg.net/text.asp?2013/38/1/17/134798


Full Text

 Introduction



Diabetes mellitus (DM) is a major medical problem worldwide. Diabetes causes an array of long-term systemic complications that have a huge impact on the patient as well as the society, as the disease typically affects individuals in their most productive years 1. Patients with diabetes often develop ophthalmic complications, such as corneal abnormalities, glaucoma, iris neovascularization, cataracts, and neuropathies. The most common and potentially most blinding of these complications, however, is diabetic retinopathy (DR) 2.

DR is associated with an increased number and size of platelet-fibrin thrombi in the retinal capillaries compared with normal 3. These thrombi can contribute toward capillary obliteration and retinal ischemia. It has been reported that chronic hyperglycemia causes an increase in the diacylglycerol levels in the retina, which may activate protein kinase C 4. Through increased intracellular Ca2+, protein kinase C stimulates endothelial cells, leukocytes, and platelets to produce platelet-activating factor (PAF) 5. PAF, confined to membranes, stimulates PAF receptors 6 on platelets, inducing the activation of these platelets. Activated platelets produce a number of platelet-derived microparticles 7, which contribute toward thrombus formation by providing and expanding a catalytic surface for the coagulation cascade. Pathological levels of fluid shear stress in abnormal retinal blood vessels affected by hyperglycemia may cause both further platelet aggregation (PA) and shedding of more microparticles from the platelet plasma membrane 8.

In addition, elevated sorbitol in the retina and erythrocytes can reduce vascular prostacyclin accompanied by an increased synthesis of thromboxane through induction of ADP 9 or collagen 10 in whole blood. The imbalance of thromboxane and prostacylin enhances platelet hyperactivity 11. Adhesion proteins that are cofactors in the aggregation of human platelets and mediating the ADP-induced response of these cells are also increased significantly 12. Although increased PA has been shown in patients with DM, its possible correlation with the severity of DR remains to be elucidated 13.

We aimed to compare PA in patients with type II diabetes with no apparent retinopathy (NAR), nonproliferative DR (NPDR), proliferative DR (PDR) patients, and healthy individuals to evaluate the possible role of PA in the pathogenesis and staging of DR and also to examine the correlation between PA and some of the predisposing factors of DR.

 Patients and methods



Patients

Thirty patients with type II diabetes were enrolled in this study. Patients were attending the Opthalmology Clinic of Ain Shams University Hospitals. They were classified into three groups: 10 with NAR, mean age 52.7±3.7 years. There were five men and five women, with a male to female ratio of 1 : 1, and 10 patients with NPDR, mean age 56.7±4.0 years. There were six men and four women, with a male to female ratio of 1.5 : 1, and 10 patients with PDR, mean age 60.2±1.8 years. There were six men and four women, with a male to female ratio of 1.5 : 1. Ten healthy individuals were studied as a control group, mean age 50.2±2.6 years. There were six men and four women, with a male to female ratio of 1.5 : 1. All patients were informed about the objectives and procedures of the study and provided written consent.

Methods

Sampling

Three milliliters of venous blood was collected in EDTA. These anticoagulated blood samples were used for complete blood count. Another blood sample was drawn directly into an evacuated plastic tube containing 3.2% buffered sodium citrate (one part anticoagulant to nine parts blood) for PA. After sample collection, proper mixing of the anticoagulant was ensured by gently inverting the tube by hand three to four times. Hemolyzed blood samples were excluded. Samples were stored undisturbed at room temperature (+16 to +26°C) and were stable for up to 4 h. Plasma samples were used for the estimation of fibrinogen levels, fasting plasma glucose (FPG), and glycated hemoglobin (HbA1c).

Criteria for the diagnosis of diabetes mellitus 14

Symptoms of diabetes plus casual plasma glucose concentration of at least 200 mg/dl (11.1 mmol/l). Casual is defined as any time of the day without taking into consideration time since the last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss.FPG of at least 126 mg/dl (7.0 mmol/l). Fasting is defined as no caloric intake for at least 8 h.Two hours postprandial blood glucose of at least 200 mg/dl (11.1 mmol/l) during an oral glucose tolerance test. The test should be performed as described by the WHO using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.

Diagnosis and staging of diabetic retinopathy 15

Complete ocular examination was carried out for all the patients, including best-corrected visual acuity using a high-contrast Snellen’s letter, slit-lamp examination (Topcon Corp., Tokyo, Japan) of the anterior segment, measurement of the ocular tension by a Goldman applanation tonometer (Haag-Streit, Berne, Switzerland). The pupils were dilated using tropicamide 1% eye drops and phenylephrine 2.5% eye drops for fundus examination using the indirect ophthalmoscope (Keeler, London, UK) with a +20 D double aspheric lens (Volk Optical Inc., Mentor, Ohio, USA) and slit-lamp biomicroscopy with the +90 D biconvex lens (Volk Optical Inc.). All the patients were subjected to fundus fluorescein angiography, where the pupils were dilated using tropicamide 1% eye drops and phenylephrine 2.5% eye drops. Fluorescein dye (10%) was used to perform the angiography using the Topcon TRX 50IX (Topcon Corp.) with the software version Imagenet 2000. The technique involved taking color pictures of the patient fundus, then taking a red-free image, followed by an intravenous injection of 5 ml of 10% sodium fluorescein, and taking the images at ∼1-s intervals.

Patients were divided into groups according to the staging of DR:

No apparent diabetic retinopathy: no abnormalities.NPDR. Mild NPDR: microaneurysms only. Moderate NPDR: more than just microaneurysms but less than severe NPDR. Severe NPDR: no signs of proliferative retinopathy, with any of the following: greater than 20 intraretinal hemorrhages in each of four quadrants; definite venous beading in two or more quadrants; prominent intraretinal microvascular abnormalities in one or more quadrants.PDR: optic disc or retinal neovascularization and/or vitreous or preretinal hemorrhage.

Platelet aggregation by platelet function analyzer

The Dade Behring PFA-100 (Marburg, Germany) is a microprocessor-controlled instrument that provides a quantitative measure of primary, platelet-related hemostasis at high shear stress 16. To perform the test, 0.8 ml of citrated whole blood was transferred into the reservoir of a disposable test cartridge within 2 h of blood sampling. The anticoagulated blood was warmed to 37°C and drawn under vacuum through a 200-pm-diameter stainless-steel capillary and a 150-pm-diameter aperture in a nitrocellulose membrane coated with collagen and adenosine diphosphate. In response to the high shear rates and the agonists, a platelet aggregate is formed that blocks blood flow through the aperture.

Interpretation of results

The time taken to occlude the aperture is reported as the closure time (CT) in seconds and is measured to a maximum of 300 s 17. The reference range was considered to be between 68 and 121 s 18.

Other laboratory tests

FPG, HbA1c, and fibrinogen levels were assessed for all the participants studied.

Statistical analysis

Statistical analysis of the data was carried out using the SPSS 15 software package (SPSS Inc., Chicago, Illinois, USA) under the Windows 7 operating system. Graphic presentation of data was carried out using EXCEL 2010 (Redmond, Washington, USA) software. Qualitative data parameters were presented in the form of frequency and percentage and were analyzed for group differences and parameter associations using χ2-test (χ2 value) according to the nature of the data. The central tendency of quantitative data parameters was presented in the form of mean and median, and the measure of spread was presented as SD, 25th, and 75th percentiles. Comparative analysis was carried out using analysis of variance (F value). Variables correlations were performed using the Pearson correlation test (r value).

The Box Plot (Box-and-Whisker Plot), the central box, represents the values from the lower to the upper quartile (25th to 75th percentile). The middle line represents the median. The rounded marker represents the mean. The upper whisker represents the highest of either the maximum value or 1.5 interquartile range distance from the 75th percentile. The lower whisker represents the lowest of either the minimum value or 1.5 interquartile range distance from the 25th percentile. Probability level (P value) was assumed to be significant if less than 0.05 and highly significant if the P value was less than 0.001. P value was considered nonsignificant if greater than or equal to 0.05.

 Results



The demographic, clinical, and laboratory characteristics of the studied groups are shown in [Table 1].{Table 1}

On comparing all the groups studied in terms of demographic data, duration of diabetes, and laboratory parameters, the mean age and duration of diabetes were significantly higher in the PDR group (P<0.001). The PDR group also showed higher levels of platelet count and HbA1c (P<0.001) [Table 2]. Both NPDR and PDR showed higher levels of fibrinogen (P<0.001) [Table 2]. The PDR group showed the shortest median of CT (P<0.001) [Table 2] and [Figure 1].{Table 2}{Figure 1}

The correlation between CT and age, duration of diabetes, and laboratory parameters showed that CT was inversely correlated with the fibrinogen level in the NAR group (P=0.000) [Table 3]. Also, CT was inversely correlated with the duration of diabetes in the NPDR group (P=0.04) [Table 4]. No correlation was found between CT and any of the studied parameters in the PDR group (P>0.05) [Table 5].{Table 3}{Table 4}{Table 5}

 Discussion



The pathophysiology of diabetic microangiopathy and macroangiopathy may depend on whether the patient has type I or II disease, but both types of diabetes are associated with a hypercoagulable state driven by platelet hyper-reactivity 19. Among the factors suggested to be involved in the development of DR is altered platelet function 20.

We studied PA in different stages of DR and in healthy individuals using a PFA-100 analyzer. PDR had the shortest CT (P<0.001) [Table 2] and [Figure 1], indicating a higher rate of PA than the control, NAR, and NPDR groups, which may imply a relation between the rate of increase in PA and stages of DR. Agardh et al. 20 found that patients with PDR had an increased PA in vitro after stimulation with collagen and ADP. It was shown before that low-dose thrombin (<0.5 µg/ml)-stimulated PA in the diabetic patients was considerably elevated compared with healthy control individuals 21.

Similarly, it was reported previously that platelets are activated in diabetes 19,22. PA was greater in patients with DR 23. A study found that the concentration of ADP that produced 50% maximum intensity of aggregation was 1.81 µmol/l in patients without DR, 0.92 µmol/l in patients with background DR, 0.85 µmol/l in patients with ischemic PDR, and 0.44 µmol/l in patients with edematous DR 24. It was shown before that light scattering can quantify spontaneous platelet aggregation. The total light intensities of aggregate size (small, medium, and large) were compared in different stages of DR. NDR showed a significant increase in small aggregates as compared with the control. Pre-proliferative diabetic retinopathy showed a tendency toward an increase in medium and large aggregates compared with the control. PDR showed a tendency toward an increase in large aggregates compared with the control. Small aggregates increased before DR became evident. Aggregate sizes increased with the progress of DR 25.

However, Tahira 26 did not find any significant difference between PA of diabetic patients with and without DR. Also, in a previous study, no significant correlation was found between PA and the severity of DR 27.

No correlation was found in this work between PA and age in any of the stages of DR [Table 3], [Table 4] and [Table 5] as mentioned before 28. In contrast, previously, it has been reported that platelet nitric oxide production and responsiveness decreases with age and this is reflected in increased circulating monocyte-platelet aggregates 29. In our study, the mean age was significantly higher in the PDR group (P<0.001) [Table 2]. It has been suggested before that the prevalence and severity of DR increase with age 30.

In this work, a significant inverse correlation was detected between CT and duration of diabetes in the NPDR group (P=0.04) [Table 4], which suggests a relation between the increase in PA and duration of disease. Contrary to our expectations, we did not find a correlation between CT and duration of diabetes in the PDR group (P=0.92) [Table 5]; this may be because of the limited number of cases studied. It has been reported before that platelet accumulation increased with the duration of diabetes 31.

The duration of diabetes in this study was significantly higher in the PDR group (P<0.001) [Table 2]. Previously, it has been reported that the severity of DR increases with the duration of diabetes 30 and that the longer the duration of diabetes, the higher the prevalence of DR 32,33.

No correlation was found in this study between CT and HbA1c in any of the stages of DR [Table 3], [Table 4] and [Table 5]. In agreement with this, Mandal et al. 34 reported that there was no relationship between PA and glycosylated Hb levels. However, Demirtunc et al. 35 studied 70 patients with type II DM and suggested a close relationship between poor glycemic control and increased platelet activity in those patients. The median HbA1c% in this study was higher in NPDR and PDR groups (7.7 and 8.7, respectively) than the NAR and control groups (P<0.001) [Table 2], which reflects the role of glycemic control in the progression of DR. A previous work showed that the frequency of DR was 46.6% (28.8% have NPDR and 17.8% have PDR). There was a statistically significant relationship between HbA1c levels and DR (both NPDR and PDR) (P<0.000). The frequency of retinopathy (both background and proliferative) was 4.8% in the group of diabetics with a mean HbA1c level less than 6%, 8.7% in those with a mean HbA1c level between 6.1 and 6.9%, 62.8% in those with a mean HbA1c level between 7 and 9.9%, and 82.2% in those with a mean HbA1c level exceeding 10% 33.

Also, HbA1c value greater than 8.0% was significantly related to sight-threatening diabetic retinopathy 36. In contrast, Singh et al. 37 reported that the mean value of HbA1c was higher in proliferative retinopathy than in background retinopathy, but with no statistical significance.

In this study, a significant inverse correlation was found between CT and fibrinogen level only in the NAR group (P=0.000) [Table 3], which may be because of our small sample size. It has been reported before that elevation of the fibrinogen level in diabetes may contribute toward fibrin clot formation and PA 38. In this study, both NPDR and PDR groups had higher levels of fibrinogen than the NAR and control groups (P<0.001) [Table 2] as reported previously 39. Previously, it was shown that the measured fibrinogen levels of patients with different stages of DR were in the pathological range and were significantly higher than those of healthy volunteers, but there was no significant difference in the fibrinogen level between the different stages of retinopathy 40.

The platelet count in this work did not correlate with PA in either stages of DR [Table 3], [Table 4] and [Table 5]. However, the PDR group showed higher levels of platelet count (P<0.001) [Table 2]. It has been shown previously that the mean platelet volume values of patients with PDR were significantly higher than the values of the control group (P<0.05). A significant correlation was found between the degree of retinopathy and the mean values of mean platelet volume in diabetic patients (P<0.05) 41. It has been found before that the mean platelet component was 26.9 g/dl in healthy individuals and 22.5 g/dl in diabetics. Patients with NPDR and PDR showed a similar decrease in the mean platelet component level, whereas those with no DR were at an intermediate position between normal controls and DR 23.

 Conclusion



PA is increased in the PDR stage in comparison with NAR, NPDR stages, and healthy individuals. This may provide a clue of its role in the pathogenesis and staging of DR. Also, this study highlights the crucial contribution of glycemic control and duration of diabetes in the progression of DR. Studies assessing the correlation of PA with glycemic control and duration of disease are recommended to be carried out on a wider scale to verify its association with the most important predisposing factors of DR and direct clinicians’ attention toward this association; if proved in controlling DR patients.

Acknowledgements

The facilities offered by the Clinical Pathology Department, Hematology Unit, Faculty of Medicine, Ain Shams University, to carry out this work are greatly appreciated.[41]

References

1Federman JL, Gouras P, Schubert H, Slusher MM, Vrabec TRPodos SM, Yanoff M. Systemic diseases. Retina and vitreous: textbook of ophthalmology. 1994;9:7–24
2Aiello LM, Cavallerano JD, Aiello LP, Bursell SEGuyer DR, Yannuzzi LA, Chang S, et al. Diabetic retinopathy. Retina vitreous macula. 1999;2:316–344
3Jian B, Jones PL, Li Q, Mohler ER III, Schoen FJ, Levy RJ. Matrix metalloproteinase-2 is associated with tenascin-c in calcific aortic stenosis. Am J Pathol. 2001;159:321–327
4Boeri D, Maiello M, Lorenzi M. Increased prevalence of microthromboses in retinal capillaries of diabetic individuals. Diabetes. 2000;50:1432–1439
5Takahashi T, Hato F, Yamane T, Fukumasu H, Suzuki K, Ogita S, et al. Activation of human neutrophils by cytokine-activated endothelial cells. Circ Res. 2001;88:422–429
6Bussolino F, Gremo F, Tetta C, Pescarmona GP, Camussi G. Production of platelet-activating factor by chick retina. J Biol Chem. 1986;261:16502–16508
7Omoto S, Nomura S, Shouzu A, Hayakawa T, Shimizu H, Miyake Y, et al. Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy. Nephron. 1999;81:271–277
8Nomura S, Nakamura T, Cone J, Tandon NN, Kambayashi J. Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation. Cytometry. 2000;40:173–181
9Li M, Goto S, Sakai H, Kim JY, Ichikawa N, Yoshida M, et al. Enhanced shear-induced von Willebrand factor binding to platelets in acute myocardial infarction. Thromb Res. 2000;100:251–261
10De la Cruz JP, Maximo MA, Blanco E, Moreno A, Sanchez de la Cuesta F. Effect of erythrocytes and prostacyclin production in the effect of fructose and sorbitol on platelet activation in human whole blood in vitro. Thromb Res. 1997;86:515–524
11Phillips AO, Morrisey K, Steadman R, Williams JD. Decreased degradation of collagen and fibronectin following exposure of proximal cells to glucose. Exp Nephrol. 1999;7:449–462
12De La Cruz JP, Moreno A, Ruiz-Ruiz MI, Garcia Campos J, Sanchez de la Cuesta F. Effect of camonagrel, a selective thromboxane synthase inhibitor, on retinal vascularization in experimental diabetes. Eur J Pharmacol. 1998;350:81–85
13Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care. 2001;24:1476–1485
14. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2004;27:s5–s10
15. ETDRS report 9. Ophthalmology. 1991;98:766–785
16Wasiluk A. The expression of vWF receptor on newborn platelets. Med Sci Monit. 2005;11:CR545–CR548
17Jilma B. Platelet function analyzer (PFA-100): a tool to quantify congenital or acquired platelet dysfunction. J Lab Clin Med. 2001;138:152–163
18 How to define and determine reference intervals in the clinical laboratory. Approved Guideline. 20002nd ed. Wayne, PA NCCLS
19Sobol AB, Watala C. The role of platelets in diabetes-related vascular complications. Diabetes Res Clin Pract. 2000;50:1–16
20Agardh CD, Agardh E, Bauer B. Platelet aggregation in type I diabetics with and without proliferative retinopathy. Acta Ophthalmol. 1987;65:358–362
21Kajita K, Ishizuka T, Miura A, Kanoh Y, Ishizawa M, Kimura M, Yasuda K. Increased platelet aggregation in diabetic patients with microangiopathy despite good glycemic control. Platelets. 2001;12:343–351
22Carr ME. Diabetes mellitus a hypercoagulable state. J Diabetes Complications. 2001;15:44–54
23Bae SH, Lee J, Roh KH, Kim J. Platelet activation in patients with diabetic retinopathy. Korean J Ophthalmol. 2003;17:140–144
24De La Cruz JP, Moreno A, Guerrero G, Ortega G, Gonzalez-Correa JA, Sanchez De La Cuesta F. Nitric oxide-cGMP and prostacyclin-camp pathways in patients with type II diabetes and different types of retinopathy. Pathophysiol Haemostat Thromb. 2002;32:25–32
25Yamamoto T, Kamei M, Yasuhara T, Tei M, Ouchi M, Komori H, et al. Platelet aggregate evaluation in diabetic retinopathy with particle counting method using light scattering. J Eye. 2003;20:1027–1031
26Tahira T. Platelet aggregation studies in diabetic retinopathy. Professional Med J. 2002;9:29–35
27Yamamoto T, Kamei M, Yokoi N, Yasuhara T, Tei M, Kinoshita S. Platelet aggregates in various stages of diabetic retinopathy: evaluation using the particle-counting light-scattering method. Graefes Arch Clin Exp Ophthalmol. 2005;243:665–670
28Güven F, Yilmaz A, Aydin H, Korkmaz I. Platelet aggregation responses in type 2 diabetic patients. Health. 2010;2:708–712
29Goubareva I, Gkaliagkousi E, Shah A, Queen L, Ritter J, Ferro A. Age decreases nitric oxide synthesis and responsiveness in human platelets and increases formation of monocyte-platelet aggregates. Cardiovasc Res. 2007;75:793–802
30Vernon SA. An overview of the eye in diabetes. J R Soc Med. 2003;96:266–272
31Yamashiro K, Tsujikawa A, Ishida S, Usui T, Kaji Y, Honda Y, et al. Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood–retinal barrier breakdown. Am J Pathol. 2003;163:253–259
32Jenchitr W, Samaiporn S, Lertmeemongkolchai P, Chongwiriyanurak T, Anujaree P, Chayaboon D, Pohikamjorn A. Prevalence of diabetic retinopathy in relation to duration of diabetes mellitus in community hospitals of Lampang. J Med Assoc Thai. 2004;87:1321–1326
33Özmen B, Güçlü F, Kafesçiler S, Özmen D, Hekimsoy Z. The relationship between glycosylated haemoglobin and diabetic retinopathy in patients with type 2 diabetes. Turk Jem. 2007;11:10–15
34Mandal S, Sarode R, Dash S, Dash RJ. Hyperaggregation of platelets detected by whole blood platelet aggregometry in newly diagnosed noninsulin-dependent diabetes mellitus. Am J Clin Pathol. 1993;100:103–107
35Demirtunc R, Duman D, Basar M, Bilgi M, Teomete M, Garip T. The relationship between glycemic control and platelet activity in type 2 diabetes mellitus. J Diabetes Complications. 2009;23:89–94
36Raman R, Verma A, Pal SS, Gupta A, Vaitheeswaran K, Sharma T. Influence of glycosylated hemoglobin on sight-threatening diabetic retinopathy: a population-based study. Diabetes Res Clin Pract. 2011;92:168–173
37Singh R, Prakash V, Shukla PK, Gautam S, Maurya OP. Glycosylated hemoglobin and diabetic retinopathy. Ann Ophthalmol. 1991;23:308–311
38Ford I, Singh TP, Kitchen S, Makris M, Ward JD, Preston FE. Activation of coagulation in diabetes mellitus in relation to the presence of vascular complications. Diabet Med. 1991;8:322–329
39Rema M, Mohan V, Snehalatha C. Role of coagulation factors in diabetic retinopathy. Int J Diabetes Dev C. 1995;15:14–16
40Vekasi J, Marton Zs, Kesmarky G, Cser A, Russai R, Horvath B. Hemorheological alterations in patients with diabetic retinopathy. Clin Hemorheol Microcirc. 2001;24:59–64
41Ateş O, Kiki İ, Bilen H, Keleş M, Koçer İ, Kulaçoğlu DN, Baykal O. Association of mean platelet volume with the degree of retinopathy in patients with diabetes mellitus. Eur J Gen Med. 2009;6:99–102